The ghrelin receptor is a typical rhodopsin receptor-like G protein-coupled receptor and was identified 1996 as the growth hormone secretagogue receptor GHS-R1a1. Although involved in the control of growth hormone secretion, the endogenous ligand ghrelin has potent orexigenic effects and stimulates food intake2,3. The major target for ghrelin is the hypothalamus, where the ghrelin receptor is mainly expressed in the NPY/AgRP neurons of the arcuate nucleus4, the center for energy homeostasis. Furthermore, a high ghrelin receptor expression is observed in the ventro-medial nucleus of the hypothalamus. This region is considered to be the regulation center for fatty acid metabolism5,6.
The ghrelin receptor possesses an exceptionally high constitutive activity. It was suggested to induce constant appetite and to trigger food intake in between meals7,8. In this context, reducing the basal activity of the ghrelin receptor could be an innovative and efficient strategy for obesity treatment9. Indeed, the increasing problem of obesity, not only in high-income states but also in low- and middle-income countries, urges the development of anti-obesity drugs10. Currently, treatments are restricted to only few pharmaceuticals, most presenting moderate benefits, poor tolerance and strong side effects11-13. Although life-style changes are mandatory for significant weight loss, bariatric surgery is considered to be the only effective treatment against morbid obesity. However, surgery presented high risks, high costs and always requires highly qualified performers14. Interestingly, the reason of sustainable weight loss after bariatric surgery is suggested to be due to a modification of gut hormone levels15. Thus, a specific inverse agonist able to reduce basal signaling of the ghrelin receptor and therefore, appetite and food intake in between meals could be in fact an efficient pharmaceutical against obesity.
A modified analog of substance P, [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P (MSP), was the first described ghrelin receptor inverse agonist (FIG. 1). MSP acts as a partial antagonist, but more importantly as an inverse agonist at the ghrelin receptor inhibiting the spontaneous, ligand-independent signaling16. Extensive structure-activity relationship (SAR) studies followed this discovery. After systematic truncation of the N-terminus of MSP, the C-terminal heptapeptide fQwFwLL-NH2 was identified as the minimal sequence able to act at the ghrelin receptor with inverse agonist properties. Affinity and activity were decreased 5.3-fold and 1.6-fold, respectively, in comparison to the full-length modified substance P. In addition, the pentapeptide wFwLL-NH2 (SEQ ID NO:1) is the minimal sequence able to bind the receptor (IC50=530±230 nM)17. This compound showed a biphasic concentration-response curve. It acts as a partial agonist at low peptide concentrations and as an inverse agonist at concentrations higher than 1 nM. Interestingly, this dual response could be modulated by modification of chemophysical properties at the N-terminus. Introduction of a positively charged amino acid (Lys or Arg) led to highly potent inverse agonists (EC50=36±8 and 21±2, respectively), whereas introduction of a neutral amino acid such as Ala revealed a highly potent agonist18. In addition, the aromatic core wFw appeared to be crucial for receptor binding17. Ala-scan of MSP demonstrated that substitution of both D-tryptophanes with L-alanine resulted in a total loss of activity and substitution of L-phenylalanine produced a 10-fold decrease in the inverse agonist activity.
There is a need for highly potent inverse agonists with high efficacy that may be useful in the treatment of feeding-related disorders. The inventors of this application surprisingly found that modification of the D-tryptophane at position 2 (‘w2’) in the peptide sequence KwFwLL-NH2 (SEQ ID NO:2) resulted in peptides which inhibited the ghrelin receptor more strongly than the basic peptide KwFwLL-NH2 (see Table 2 and FIG. 3). That is, the modified peptides had a higher efficacy. It was further found that the phenylalanine is essential for inverse agonistic activity (see Table 5 and FIG. 6) and that modification of D-tryptophane at position 4 (‘w4’) in the peptide sequence KwFwLL-NH2 (SEQ ID NO:2) resulted in peptides having agonistic properties.